endothelin-1 and ebselen

Experiments were designed to test the hypothesis that elevated levels of endothelin 1 (ET-1) in the vasculature activate NADPH oxidase and/or uncoupled nitric-oxide synthase (NOS), resulting in O2-* production, and mediate increased constriction. Rat aortic rings were incubated with ET-1 or vehicle in the presence and absence of superoxide dismutase (SOD), ebselen (glutathione peroxidase mimetic), apocynin (NADPH oxidase inhibitor), L-NAME (Nomega-nitro-L-arginine methyl ester) (NOS inhibitor), tetrahydrobiopterin (BH4) (NOS cofactor), or selective ETA and ETB receptor antagonists (BQ-123 [cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)] and A-192621 [[2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid]], respectively). O2-* production was monitored by oxidized dihydroethidine staining and/or lucigenin chemiluminescence. ET-1 significantly increased O2-* production compared with vehicle. SOD, ebselen, and apocynin inhibited the ET-1-induced increase in O2-* in intact and endothelium-denuded aorta. L-NAME and BH4 inhibited the ET-1-induced increase in O2-* in intact tissue, whereas these two compounds had no effect on ET-1-induced O2-* in endothelium-denuded aorta. Preincubation with BQ-123 or A-192621, individually, had no effect on ET-1-induced O2-*; however combining both antagonists inhibited the ET-1-stimulated increase in O2-*. Rat aortic rings were incubated with ET-1 or vehicle in the presence or absence of sepiapterin (BH4 synthesis substrate) or apocynin and mounted on wire myographs to determine isometric force generation in response to increasing KCl concentrations. ET-1 increased the contractile response to KCl compared with vehicle. Treatment with either sepiapterin or apocynin attenuated the ET-1-mediated increase with no effect of sepiapterin or apocynin alone. These data support the hypothesis that ET-1 increases vascular tone, in part, through ETA/ETB receptor activation of O2-* production from NADPH oxidase and NOS uncoupling.

Topics: Acetophenones; Animals; Aorta; Azoles; Biopterins; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Isoindoles; Luminescence; Luminescent Measurements; Male; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organoselenium Compounds; Pyrrolidines; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Superoxides; Vasoconstriction

To evaluate the possible role of the antioxidant ebselen in the treatment of cerebral vasospasm, we examined the effects of ebselen on the vasoactive mechanisms induced by endothelin (ET)-1, oxyhemoglobin, and oxygen-derived radicals.. Isolated rabbit basilar arteries with intact endothelium were fixed in a perfusion system and perfused intraluminally. Contraction of the artery was detected as an increase in perfusion pressure.. Ebselen, in a certain concentration range (3 x 10(-6) and 10(-5) mol/L), significantly reduced the contractile response to ET-1 (10(-10) to 10(-8) mol/L) but not the contraction induced by 40 mmol/L potassium. It reduced the contraction induced by 10(-4) mol/L 1,2-dioctanoyl-sn-glycerol, a protein kinase C activator. Addition of 10(-5) mol/L dithiothreitol, a sulfhydryl-reducing agent, partially reversed the inhibitory effects of ebselen on ET-1- and 1,2-dioctanoyl-sn-glycerol-induced contractions. Ebselen (10(-5) mol/L) as well as a combination of catalase (1000 units/mL) and superoxide dismutase (150 units/mL) inhibited the potentiating effects of oxyhemoglobin (10(-5) mol/L) on ET-1-induced contraction. Both ebselen and catalase inhibited the contractile response to hydroxyl radical generated by ferrous ion (10(-3) mol/L) plus hydrogen peroxide (10(-2) mol/L). Ebselen reduced the response to potassium when a high dose (3 x 10(-5) mol/L) was applied and failed to preserve contractility of the preparation after exposure to hydroxyl radical.. Ebselen suppressed ET-1-induced contraction and synergetic interaction between oxyhemoglobin and ET-1, where free radical formation was involved. These effects may result from modification of the intracellular regulatory system including protein kinase C, as well as from protection against free radicals.

Topics: Animals; Antioxidants; Azoles; Basilar Artery; Diglycerides; Dithiothreitol; Drug Synergism; Endothelin-1; Hydroxyl Radical; In Vitro Techniques; Isoindoles; Male; Organoselenium Compounds; Osmolar Concentration; Oxidoreductases; Oxyhemoglobins; Perfusion; Potassium; Rabbits; Sulfhydryl Reagents; Vasoconstriction; Vasoconstrictor Agents